The endogenous polyamines, in particular, putrescine (1,4-butanediamine), spermidine (N-[3-aminopropyl]-1,4-diaminobutane) and spermine (N,N'-bis-[3-aminopropyl]-1,4-butane-diamine) have been the subject of increasing research, due to the growing appreciation of their physiological importance. These polyamines have regulatory roles in tissue growth and interact with nucleic acids, as well as exhibiting a variety of effects upon macromolecular synthesis, expression and metabolism (Tabor et al., Ann. Rev. Biochem., 30, 597 (1961); Janne et al., Biochem. Biophys. Acta, 473,241, (1978); and Tabor et al., Ann. Rev. Biochem., 45,285 (1976)).
Accounts on the biochemistry, regulation and potential functions of polyamines may be found in recent review articles (Tabor et al., Ann. Rev. Biochem., 53,749 (1984); Pegg et al., Ann. J. Physiol., 243, C212 (1982); Pegg, Biochem J., 234, 249 (1986); Seiler et al., Acta Biochem. Biophys. Hung, 23, 1 (1988); and, Seiler et al, Int. J. Biochem., 22, 211, (1990)) from which most of this background discussion is excerpted.
Polyamine homeostasis and adjustment of the intracellular concentrations to physiological requirements are regulated by both synthetic and catabolic reactions. De novo biosynthesis can in principle be substituted by polyamine transport from the environment.
In cells lacking amine oxidases which oxidatively deaminate polyamines, the release of intracellular polyamines is essential for the regulation of polyamine cellular concentrations. Currently available evidence suggests that N.sup.1 -N.sup.2 -diacetylspermine may also fulfill regulatory requirements. The actual signals which control polyamine release are not known. However, there exists strong evidence in the scientific literature that the proportion of N'-acetylspermine and N'-acetylspermidine which remains intracellular and which is oxidatively cleaved into spermidine and putrescine is controlled by outward transport (Seiler, J. Physiol. Pharmacol., 65, 2034 (1987)).
In vivo studies involving the metabolism of labeled polyamines have shown that polyamines may be taken up by tissues from the circulation. It is further well known that some tissues exhibit a high demand for polyamines. Examples of such tissues include the prostate (Clark et al., J. Nucl. Med., 16, 337 (1975)), tumor cells (Volkow et al., Science, 221,673 (1983)) and normal but rapidly proliferating cells (Janne et al., Acta Chem. Scand., 20, 1174 (1966); Seiler et al., Brain Res., 22, 81 (1970)). Enhanced uptake of .sup.14 C-labeled polyamines by tissues with depleted polyamine stores has repeatedly been observed (Chaney et al., Life Sci., 32, 1237 (1983); Huston et al., Cancer Res., 44, 1034 (1984)).
Recently, much research has been undertaken to characterize and understand the particular transporters which control polyamine uptake in mammalian cells. Early work on polyamine transport related to bacteria and other microbia and is reviewed in Tabor et al., Pharmac. Rev., 16, 245 (1964) and Bachrach, "Function of Naturally Occurring Polyamines," pp. 51-52, Academic Press, N.Y. (1973). Other papers describing polyamine transport in specific microbia are also available in the literature (Davis et al., Arch. Biochem. Biophy., 267,479 (1988) and Kashiwagi et al., J. Bact., 165, 972 (1986)).
Polyamine uptake systems in mammalian cells resemble the uptake systems of amino acids. Accordingly, most researchers studying polyamine transport have used research strategies which have had prior success in characterizing amino acid uptake.
To date there still exists no complete understanding of polyamine transport systems in mammalian cell lines. However, from what is disclosed in the literature, it appears that polyamine transport is energy and temperature dependent, and saturable. Therefore, it seems that it is a carrier-mediated transport system. An exception is polyamine transport into perfused rabbit lung or lung slices which appears not to be energy dependent or Na.sup.+ activated, suggesting that polyamine uptake into the lungs of this species may instead occur via diffusion (Rao et al., Biochem. Biophys. Acta, 966, 22 (1988)) rather than an active carrier system. Polyamine uptake in lungs of other species, including rats, is saturable and energy-dependent (Rannels et al).
Available evidence suggests that macromolecular synthesis of RNA and promins is required to adapt polyamine transport to enhanced polyamine requirement (Martin et al., Int'l. Symp. Polyamines in Biochem. Clin. Res., Sorrento (Italy), Abst. p. 67 (1988). Most cells appear to have a single transporter for putrescine, spermidine and spermine as shown by competitive studies. However, some cells appear to have more than one pathway for polyamine uptake wherein each transporter comprises a different affinity for putrescine, spermidine and spermine (Kumagai et al., Am. J. Phys., 254, G81 (1988); Minchin et al., Int'l. Symp. Polyamines Biochem. Clin. Res,, Sorrento (Italy), Abst. p. 67 (1988); Byers et al., Am. J. Physiol., 252, C663 (1987); Feige et al., Biochem. Biophys. Acta, 846, 93 (1985); and De Smedt et al., Biochem. Biophys. Acta, 1012, 171 (1989)).
The affinity for the carrier increases from putrescine to spermidine and spermine with published K.sub.m values in the low .mu.M range. Polyamine transport is observed in the absence of sodium; however, the increase of sodium to physiological concentrations (116 mM) usually increases the transport rate by 60%. Dissipation of the sodium gradient by ionophores has been shown to inhibit the sodium-dependent portion of polyamine uptake.
While high affinity sodium-activated transport is usually observed, some cell types exhibit sodium-independent polyamine uptake. For example, in LLC-PK1 renal epithelial cells both the sodium-activated and the sodium-independent transport systems were saturable. By contrast, in adrenocortical cells the sodium independent transport system was not saturable and preferentially transported spermine (Feige et al., Biochem. Biophys. Acta, 846, 93 (1985)).
Activation of polyamine transport into mammalian cells by thiols (glutathione, dithiothreitol) and inhibition by thiol reagents (p-chloromercuribenzene sulfonate, N-ethylmaleimide) has been studied by many researchers. The results obtained by these researchers suggest that the sodium-activated transporter requires thiol groups to maintain active conformation.
The amino acid uptake pathways are generally regarded as different from polyamine transporters systems. However, a number of amino acids (asparagine, glutamine, serine, alanine and .alpha.-(methylamino)isobutyric acid) stimulate at 0.5 mM concentration putrescine uptake into a neuroblastoma cell line (Rinehart et al., J. Biol. Chem., 259, 4750 (1984)) and that some basic amino acids exhibit a very weak inhibitory effect on polyamine uptake.
The polyamine transporter does not appear to be specific for putrescine, spermidine or spermine. Therefore, it is likely that polyamine analogs are transported by the same system. Some researchers have compared a series of homologous diamines and triamines in relation to their ability to inhibit competitively the uptake of labelled putrescine, spermidine and spermine by L1210 leukemia cells. 1,7-Heptene diamine followed by 1,8-octanediamine and 1,6-hexanediamine were most potent among the diamines, while triamines were generally more effective as uptake competitors. Maximum inhibition was achieved using amines having comparable chain lengths to spermine and spermidine. This is consistent with the understanding in the art that the recognition site of the polyamine transporter contains at least three negatively charged groups in a distance corresponding to the distance between the positively charged nitrogen atoms of spermidine, presumably in its most stable all-trans configuration. (Gordon-Smith et al., Biochem. Pharmacol., 32, 3701, (1983)).
The primary amine groups of the polyamines seem to be essential for uptake. For example, N-alkyl substituents on the terminal amino groups of putrescine, spermidine or spermine decrease the ability of these compounds to competitively inhibit uptake. Also, substituents (F, Cl, OH, CH.sub.3) in the last two positions of the carbon chain of putrescine reduce polyamine uptake.
Methylglyoxal-bis(guanylhydrazone) (MGBG), an antileukemic drug which inhibits S-adenosylmethionine decarboxylase and which exhibits a structure highly similar to spermidine shares the polyamine transport system. The potent cytotoxicity of this compound thereby afforded researchers a selection method for identifying Chinese hamster ovary cells incapable of MGBG and polyamine transport (Mandel et al., J. Cell. Physiol., 97, 335 (1978). Genetic studies indicate that more than one gene locus is involved in MGBG uptake by these cells (Heaton et al., J. Cell. Physiol., 136, 133 (1988)).
Also, it is believed that such polyamine transport mutants should play a major role in identifying genes involved in polyamine transport, for studying the regulation of transport, and for studying the role of polyamine transport in proliferating cells and in some related disease conditions such as cancer.
For example, Ask et al. recently studied increased survival of L1210 leukemic mice by preventing utilization of extracellular polyamines using an L1210 leukemic cell line (L1210-MGBG.sup.r) deficient in polyamine transport. Their results indicated that leukemic mice bearing the polyamine uptake mutant cells exhibited a cure rate of 33% when polyamine synthesis was blocked by DFMO. By contrast, mice having non-mutant polyamine transport L1210 cells only had a two day increase in survival time after similar DFMO treatment (Ask et al., Cancer Lett., 66, 29 (1992)).
The polyamine uptake system has been implicated in the uptake of other drugs as well. For example, paraquat (N,N'-dimethyl-4,4'-dipyridylium) has been shown to inhibit the uptake of MGBG, putrescine and spermidine, but with no apparent effect on spermine uptake (Smith et al., Biochem. Pharmac., 30, 1053 (1981)).
Also, some researchers have attempted to design cytotoxic drug conjugates which are similar in structure to endogenous polyamines so that they are transported by the polyamine transport system. For example, Holley et al. recently synthesized a series of 2- and 5-nitroimidazolepolyamine conjugates which were apparently transported by the polyamine transport system. Of the compounds tested, the 2-nitroimidazole-polyamine conjugates were the most potent inhibitors of spermidine uptake (Holley et al, Biochem. Pharmacol. 43,763, (1992)).
Also, several bipyridinium, tetrapyridinium and hexapyridinium quaternary salts have been found to be potent inhibitors of putrescine uptake into B16 melanoma cells which have previously treated with difluoromethylornithine, with the potency of inhibition being apparently directly related to the number of quaternary centers (Minchin et al., Biochem. J., 262, 391 (1989).
Some inhibitors of polyamine uptake have been discovered which are structurally unrelated to polyamines. For example, the cationic peptide melittin and another calmodulin antagonist, 1.3-dihydroxy-1-(4-methyl)-4H,6H-pyrrolo[1,2-9][4,1]-benzoxazepin-4-yl)met hyl]-4-piperidinyl]-2H-benzimidazol-2-one (CG 59373B) inhibits putrescine uptake into human prostate cells. Trifluoropiperazine, a calmodulin antagonist and protein kinase C inhibitor also inhibits polyamine uptake as does the protein kinase C inhibitor H7 (1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine). Also, treatment of leukemia cells with phorbyl esters activates proteinase kinase C and has been shown to enhance spermidine uptake into murine leukemic cells. It therefore appears that protein kinase C may play a role in polyamine transport.
Various factors are believed to affect the uptake of polyamines into mammalian cells including (i) availability of polyamines in vivo, (ii) intracellular polyamines, (iii) the sialic acid coating on the cell membrane, and (iv) the growth state of the particular cells.
The availability of polyamines in the circulation is believed to be important in polyamine transport. Putrescine and spermine blood concentrations are low in comparison to spermidine (Claverie et al., Anticancer Res., 7, 765, (1987)). This is surprising given that the electrostatic binding of spermine to negatively charged groups is higher than for spermidine and putrescine. Accordingly, one would expect the concentration of free spermine to be extremely low, particularly in light of the fact that red blood cells have a negative surface charge and efficiently bind polyamines. Further evidence of the importance of spermidine uptake is the fact that the V.sub.max of spermidine is higher than that of spermine, despite its lower affinity to the transporter.
The level of intracellular polyamines is also believed to affect polyamine uptake. Depletion of intracellular polyamines has been found to generally enhance polyamine uptake several-fold. As in the case of circulating polyamines, the concentration of free intracellular polyamines is probably involved in regulation of uptake.
As noted above, the sialic acid coating on cell membranes is believed to have some effects on polyamine transport. The removal of N-acetylsialic acid from the surface of murine leukemia cells by incubation with neuraminidase enhances spermidine uptake. Re-sialated cells regain normal transport rates (Khan et al., Biochem. Arch., 5, 161 (1989)). It is believed by some researchers that the sialic cell surface coating nonspecifically binds positively charged polyamines via the carboxyl group of the sialic acid residues thereby diminishing effective concentration of transport substances at the transporter site.
Polyamine transport is also believed to be important for the ability of cells to adapt to changes in polyamine requirements. Enhanced cell growth rates are usually accompanied by both enhanced intracellular de novo synthesis and enhanced uptake of polyamines.
Trophic stimuli, including hormones and growth factors, have been observed to enhance polyamine transport rates (Pohjanpelto, J. Cell. Biol., 68, 512 (1976), DiPasquale et al., Exp. Cell. Res., 116, 317 (1978)). In these studies stimulation of human fibroblasts to cell proliferation by serum or epidermal growth factor was followed by an 18-100 fold increase of ornithine decarboxylase activity.
Also, stimulation of mouse mammary glands with insulin caused the increase of V.sub.max for spermidine and prevented effects thereof (Kano et al., J. Biol. Chem., 251, 2795 (1976)). However, it is unknown whether the hormone induced formation of the carrier or decreased carrier inactivation.
Some studies have shown that transformed cells transport polyamines better than their normal counterparts. However, in at least one study involving EJ2-ras transfected cells polyamine turnover was enhanced but transport rates remained unaffected. In contrast, N-myc transfection enhanced polyamine uptake with no affect on biosynthesis (Chang et al., Biochem. Biophys. Res. Comm, 157, 164 (1988). Thus, there exists circumstantial evidence that polyamine uptake and synthesis may be regulated by different genes. Perhaps not unexpectedly, cells exhibiting reduced growth rates have been observed to decrease polyamine uptake. For example, cells approaching cell confluency because of high cell density decrease polyamine uptake. Also, when cells are induced to differentiate and therefore reduce their rate of proliferation, their demand for polyamine has been found to diminish.
As discussed above, cellular concentrations of polyamines are regulated by at least two important pathways. The first, and best understood, involves de novo biosynthesis. The rate of cellular polyamine synthesis is governed by the activity of the enzyme ornithine decarboxylase (ODC) which catalyzes the conversion of the amino acid ornithine to the diamine putrescine. Putrescine, in turn, is then convened sequentially to spermidine and spermine by two other enzymes. The role of ODC in regulation of cell polyamine contents and its potential suitability as a pharmacological target has been facilitated by the development of DFMO, (.alpha.-difluoro-methylornithine), a highly selective and potent inhibitor of this enzyme. In some instances, inhibition of ODC with DFMO is highly effective in depleting cell polyamine cell contents and arresting cell proliferation. However, in other instances the cells seem to use compensatory pathways to maintain adequate polyamine concentrations. Results of human clinical trials with DFMO have, in general, been disappointing. Though the drug is beneficial in treating some non-neoplastic diseases (e.g., infectious conditions), its therapeutic efficacy in cancer appears to be limited.
The reason for the poor results with DFMO is believed to be attributable to the polyamine transport system of cells. As discussed supra, along with de novo polyamine synthesis, cells are apparently capable of regulating and accumulating polyamines across their plasma membrane via an energy-dependent transport system. Work by the present inventors and others suggests that most cells express at least two pathways for polyamine uptake. The first comprises a nonselective pathway for putrescine, spermidine and spermine and the second comprises a pathway which is selective for spermine and spermidine. Both pathways are markedly increased when ODC activity is depressed, either by pathologic changes or by treatment with DFMO, and are thereby able to compensate for decreased de novo polyamine synthesis. Because there exist no pharmacological agents which selectively inhibit polyamine transport, the suitability of this pathway as a pharmacological target has as yet not been adequately established.
As discussed above, most previous attempts to design inhibitors of polyamine transport have focused on polyamine analogues with a few exceptions involving several calmodulin antagonists and protein kinase C inhibitors. However, the previously examined molecules have either lacked specificity and/or exhibited unacceptable toxicity. Thus, there still exists a substantial need in the art for effective polyamine transport inhibitors which selectively inhibit the transport of specific polyamines while not exhibiting unacceptable toxicity and while not affecting the transport of other transported substances. Also, there exists a need in the art for a convenient and reproductive method for obtaining transport inhibitors of desired substances.